The present invention relates to the use of cellular chromosomal replicases of the three-component type in bioinformatic techniques such as the sequencing and amplification of DNA. The three components of these DNA polymerases are 1) a catalytic component with polymerase activity, 2) a circular protein that functions as a sliding clamp to tether the polymerase component to DNA for processivity, and 3) a clamp loader that assembles the sliding clamp onto DNA for high processivity. The clamp endows the polymerase with enhanced catalytic efficiency.
DNA Sequencing
In general, two techniques have been traditionally used to sequence nucleic acids:
The Sanger technique, also known as the dideoxy sequencing method, is used for determining the unknown nucleotide sequence of a particular nucleic acid. The DNA to be analyzed must first be isolated into a single-stranded form in order to serve as a template for the synthesis of complementary DNA. The template is hybridized to a primer and then incubated with a mixture of the four deoxyribonucleoside triphosphates and small amounts of a single chain-terminating 2xe2x80x2,3xe2x80x2-dideoxynucleoside triphosphate (lack a 3xe2x80x2-OH group) in the presence of the enzyme DNA polymerase. DNA polymerase is used, because it has the ability to synthesize a complementary copy of a single-stranded DNA template and it can also use 2xe2x80x2,3xe2x80x2-dideoxynucleoside triphosphate as a substrate.
The analogs become incorporated at the growing ends of the DNA chains resulting in chain termination since they cannot form phosphodiester bonds with incoming precursors. Thus, in the absence of the hydroxyl group, the DNA fragment is no longer a substrate for chain elongation and the growing DNA chain is terminated. The DNA synthesis is carried out in the presence of the four deoxyribonucleoside triphosphates, one or more of which is labeled with 32P, in four reaction mixtures each of which contains one of the dideoxy compounds. Upon completion of each reaction, the final reaction mixture will contain a series of fragments of new DNA, each having a common 5xe2x80x2-end but varying in length to a base-specific 3xe2x80x2-end. Following synthesis, the reaction products are separated from the template by denaturation and separated by electrophoresis. The positions of the fragments on the gel are visualized by autoradiography and the DNA sequence read directly from the autoradiogram.
The Maxam-Gilbert sequencing method uses chemical reagents that react with specific bases to break DNA preferentially at specific sites. First, the strands of the target DNA molecule are labeled with 32P at one end and the two strands are separated so that one can be sequenced. Next, the single-stranded DNA strand to be sequenced will be treated with four different chemical reagents that specifically react with one of the four bases causing a break in the strand at one or two specific nucleotides. A specific amount of chemical treatment is used so that at most a single residue of the susceptible bases(s) in the molecule will react. The reaction will yield a product labeled at the 5xe2x80x2 end with 32P and terminating at the point of cleavage.
Gel electrophoresis is used to resolve the products of each reaction by size. The pattern of radioactive bands seen on the X-ray reveals the DNA sequence. By correlating the appearance of fragments of specified length with the specific base destroyed by chemical attack, the exact order of bases along the original unbroken DNA strand can be determined.
The Polymerase Chain Reaction
A targeted DNA sequence can be selectively and repeatedly amplified in vitro by use of the PCR method. An advantage of this method is that it requires only a small quantity of the DNA that is to be amplified. Restriction enzymes are used to cut the DNA into segments, and the segments are denatured into single strands. Two flanking oligonucleotide primers complementary to the 3xe2x80x2 ends of the DNA are required at the ends of the DNA segment to be amplified. The probe is added in significant excess to the denatured DNA at a temperature between 50xc2x0 C. and 60xc2x0 C. The probe hybridizes with its correct site on the DNA and serves as a primer for DNA chain synthesis, which begins upon addition of a supply of deoxynucleotides and the temperature resistant Taq DNA polymerase. After annealing, the primers 3xe2x80x2 ends face each other as a result of having complementary opposite strands.
The three steps involved in a PCR reaction are: 1) denaturation of the original double-stranded DNA sample at a high temperature; 2) annealing of the oligonucleotide primers to the DNA template at low temperature (e.g., about 37xc2x0 C.); and 3) extension of the primers using DNA polymerase. The DNA polymerase that is currently used is Taq DNA polymerase which is thermostable and unaffected by the denaturation temperature. Each set of three steps is a cycle which is repeated many times in a PCR process. The extension products of one primer provide a template for the other primer in a subsequent cycle so that each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. The result is an exponential accumulation of the specific target fragment to approximately 2n, where n is the number of cycles.
The present invention is directed to improved processes of sequencing and amplifying DNA.
The present invention relates to a method for amplifying a nucleic acid molecule comprising subjecting the nucleic acid molecule to a polymerase chain reaction process utilizing a three component polymerase.
Another aspect of the present invention relates to a method for sequencing a nucleic acid molecule which includes subjecting the nucleic acid molecule to a nucleic acid sequencing process with a three component polymerase.
The invention further provides kits for amplifying and sequencing a DNA molecule.
The methods and kits of the present invention, utilizing compositions comprising a three component polymerase, such as DNA polymerase III (xe2x80x9cPol IIIxe2x80x9d) type enzyme from a single cell organism, provide a number of advantages over traditional PCR-based methods using other polymerases. In particular, the three component polymerase of the present invention permits a reduction in time required for nucleic acid amplification and sequencing. Because DNA Pol III-type enzymes have a faster rate of nucleotide incorporation than the repair-type enzymes commonly used in these techniques, for a 1-5 kb sequence, the protocol for amplification or sequencing can be reduced from several hours (or 10-20 hours in the case of long PCR on nucleic acid molecules 5-40 kb in size) to less than one hour, resulting in a reduction in thermal damage to the template nucleic acid molecule. Because the amplification and sequencing reactions utilizing DNA Pol III-type enzymes proceed more rapidly, the template is exposed to high temperatures for shorter periods of time. This reduction of thermal damage is particularly important in long PCR, where the ultimate success of the procedure is dependent upon a minimization of thermal damage to the target nucleic acid molecule, particularly during later cycles. The present invention should improve yields for sequences up to 50 kb in length, and, for the first time, enable the amplification of sequences of 100 kb or larger. In addition, the relatively long times required for traditional PCR-based amplification and sequencing protocols using repair-type enzymes tends to promote the formation of short, nonspecific reaction products (e.g., truncated copies of the template molecule). Rapid extension of the correct template sequence, afforded by the present invention, will reduce the number of false priming interactions that occur and, thus, provide an improvement in the specificity of the reaction. The ability of DNA Pol III-type enzymes to synthesize long sequences without dissociating from the template nucleic acid molecule, via the methods of the present invention, will improve success in long PCR by allowing nucleic acid synthesis through secondary structures and regions of high GC content in the template which tend to retard or inhibit synthesis in reactions catalyzed by the repair-type enzymes.